The Tumor Growth Inhibitory Effect of a Standardized Extract of Cultured Lentinula edodes Mycelia Using Patient Derived Xenograft Model

Abstract

Patient derived xenograft (PDX) is a powerful tool to confirm pharmacological efficacy in non-clinical studies for the development of various drugs including anti-cancer agents and therapeutic research. A standardized extract of cultured Lentinula edodes mycelia, a product name AHCC^® is produced by Amino Up Co., Ltd. (Sapporo, Japan). In this study, we investigated the inhibitory effect of AHCC^® on the growth of tumor PDX in Super SCID (severe combined immunodeficiency) mice. Effects of AHCC^® and BCG administration on the growth of renal cancer PDX implanted in Super SCID mice were evaluated by PDX growth curve. Tendency for the effects on the growth of renal cancer PDX in Super SCID by administration of AHCC^® and BCG before implanting the PDX were demonstrated. The effects of the oral administration of AHCC^® on the growth of renal, invasive and non-invasive breast cancer PDX in Super SCID mice were studied. In Super SCID mice transplanted with renal cancer PDX, AHCC^® significantly suppressed tumor proliferation from the day 48 to 83 after transplantation. In two types of breast cancer PDX, tendency of the growth inhibitory effects of AHCC^® were shown by PDX growth curve. Significant inhibitory effect was found at only one time point for during proliferation in each PDX. Super SCID-PDX model has the potential to be a useful tool to investigate for the effect of functional foods.

INTRODUCTION

We have established a number of patient derived xenograft (PDX) created by transplantation of human tissue into severe combined immunodeficient (SCID) mice. Those human tissues we used were unavoidably removed or harvested for therapeutic or diagnostic purposes at medical institutions and were to be discarded. Tumor PDX maintained its original characteristics and the graft yields many tissue fragments by proliferation in SCID mouse. Therefore, a significant number of studies is possible using almost identical cancer tissue.¹⁾ Thus, SCID-PDX model is a valuable tool for confirming pharmacological efficacy in non-clinical studies for the development of various drugs including anti-cancer agents and therapeutic research.²⁾

The immunodeficient mice that we mainly use for PDX, are originally from C.B17 SCID mice discovered by Bosma et al., Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, U.S.A.³⁾ This mouse strain was provided to Nomura by Bosma, and Nomura et al. improved it at Osaka University. The improved SCID mouse is known as Super SCID mouse⁴⁾ due to the undetectable level of serum immunoglobulin G (IgG) and IgM by enzyme-linked immunosorbent assay (ELISA), and these characteristics make it suitable for xenotransplantation. The advantage of the Super SCID-PDX model is that cancerous and noncancerous tissues and human cells are maintained for a long period of time in Super SCID mouse. In particular, cancer tissue grow and proliferated cancer tissue are more similar to clinical results. Those other characteristics also make it possible to obtain PDX on a tissue-by-tissue basis.^5,6)

The Super SCID-PDX model was initially developed to assess various toxicities for chemicals and ionizing radiation as reported by Nomura^7–9) and has also been used in the development of marketed drugs. Recently, our gastric cancer PDX was used in preclinical studies for trastuzumab deruxtecan, a treatment for HER2-positive gastric and breast cancer, and the drug was launched in 2020.^10,11) Other non-clinical trial using our PDX was gastrointestinal stromal tumor (GIST) PDX.¹²⁾ In this study, we examined whether PDX is also helpful in evaluating the suppressive effects on tumor growth using functional food.

Mushroom extracts, such as shiitake and agaricus, made from cultivated or cultured basidiomycetes, have been reported for various effects, namely immunomodulating and anti-inflammatory. These extracts contain polysaccharides, e.g., α-D-glucan, β-D-glucan, peptidoglycans, and lipopolysaccharides, which are presumed to be these functional components.¹³⁾

AHCC^® is a standardized extract of cultured Lentinula edodes mycelia, produced by Amino Up Co., Ltd. (Sapporo, Japan), and is commercially available worldwide as an immunomodulating functional food. AHCC^® is rich in partly acetylated alpha-1,4-glucan, which has been reported to be effective against a variety of diseases, including infectious diseases,^14,15) cancer, and colitis.¹⁶⁾ Its effects are related to its immunomodulating action, and it is described to activate NK cells and macrophages in animal and human experiments.^17,18)

Tumor growth and progression of human urinary tumors including renal cancer, are reported to involve individual immune system.¹⁹⁾ BCG, a live vaccine, is used as one of the standard treatments for bladder cancer in clinical practice due to the characteristic for the activation of macrophages in the same way as AHCC^®.^17,20–23) However, BCG therapy causes side effects including painful urination, frequent urination, and fever in more than 90% of patients.²⁴⁾ On the other hand, AHCC^® has no such side effects at normal intake levels and is distributed as a functional food.¹⁴⁾

An application of AHCC^® has been reported that AHCC^® is useful for the recuperation of cancer patients or their prognosis,²⁵⁾ namely reducing the side effects of chemotherapeutic agents during cancer treatment,^26–29) preventing cancer recurrence,³⁰⁾ improving the QOL prognosis, and reducing oxidative stress.³¹⁾

In this study, we found significant inhibitory effect of AHCC^® on the growth of renal cancer PDX and tendency of the inhibitory effect on the growth of breast cancer (invasive and non-invasive ductal carcinoma) PDX in Super SCID mice. In addition, BCG tended to suppress the growth of renal cancer PDX in Super SCID mice.

MATERIALS AND METHODS

PDX

Human right clear cell renal cell carcinoma (renal cancer) PDX (K928), invasive ductal carcinoma PDX (M1265) and non-invasive ductal carcinoma PDX (M502) were established and maintained as cryopreserved. A part of established renal cancer PDX without cryopreserved was transplanted to SCID mice several passages. This PDX was transplanted into SCID mice to increase the volume enough for the experiment. After thawing of the cryopreserved invasive and non-invasive ductal carcinoma PDXs, those PDXs were transplanted into a few SCID mice to expand PDXs for the experiments.

The PDXs of renal cancer, invasive ductal carcinoma and non-invasive ductal carcinoma were originated from a Japanese 79-year-old female renal cancer, a 36-year-old Japanese female breast cancer and a 77-year-old Japanese female breast cancer, respectively. The Ethics Committee at NIBIOHN approved the study protocol (Approval Numbers: 114, 219) and Osaka Police Hospital approved the study protocol (Approval Numbers: 140, 1270, 1634). All the patient tissue and informed consent from the patients were obtained by Katsuhide Yoshidome, Osaka Police Hospital. After these patient tissues were anonymized, those tissues were provided from Osaka Police Hospital to Taisei Nomura, NIBIOHN.

AHCC^®

The AHCC^® (Lot: S16-0809-2) were kindly provided by Amino Up Co., Ltd. In experiments with human mesothelioma cell lines, 2.0% AHCC^® was dissolved in 0.9% saline and this solution was intraperitoneally (i.p.) administered to be 0.2 mg per mouse body weight (BW). In the experiment with renal cancer PDX, 0.5% AHCC^® dissolved in sterile water was prepared as drinking water. In the experiment with breast cancer PDX, AHCC^® 0.5 and 2.0% AHCC^® in feed were preprepared as shown below. AHCC^® was mixed with the powdered feed CRF-1 (sterilized by ⁶⁰Co at 30kGy, Charles River Japan, Kanagawa, Japan) in Milli-Q water (Merck Millipore, Germany), formed and dry-heat sterilized (100 °C, 2.5–3h).

BCG

The BCG (Lot: KH209) was kindly provided by Japan BCG Laboratory (Tokyo, Japan). BCG (2 mg/mL) was dissolved in 0.9% saline, and this solution was i.p. administered to be 10 µg per g mouse BW.

The Super-SCID Mice

C.B17-scid/ +  male and female mice were provided by Bosma in 1986 and C.B17-scid/scid mice were improved by selective sibling mating by serum IgG and IgM below the detection limits (<1 µg/mL) by ELISA. Thus C.B17-scid/scid reduced the incidence of leukemia. C57BL/6J-scid/scid (B6-scid) and C3H/HeJ-scid/scid (C3H/HeJ-scid) mice used in the experiment were prepared by mating as described below. A C.B17-scid/scid male (N₁F₃) was mated with a C57BL/6J female. Progeny was crossed, and C57BL/6J scid homozygote mouse was repeatedly back-crossed to C57BL/6J in order to make the congenic strain of C57BL/6J-scid/scid. The same mating scheme using a C3H/HeJ female instead of a C57BL/6J female was applied to make C3H/HeJ-scid/scid congenic strain. These two congenic strains of C57BL/6J-scid/scid and C3H/HeJ-scid/scid were made by Nomura. Mice were maintained in SPF with lighting from 4:30–20:00 at 23 ± 1 °C, 50–70% humidity, bred with sterilized CRF-1 feed and filtered (Edstrom, U.S.A.) acidic water. The serum IgG and IgM were measured by ELISA at 4–6 weeks after birth. All the animal experiments were carried out in the barrier section of the National Institute of Biomedical Innovation following the guidelines for animal experimentation and anesthesia of mice was carried out using an inhalation anesthetic system (DS Pharma Biomedical Co., Osaka, Japan) with isoflurane (Mylan Pharmaceuticals Co, Osaka, Japan). The animal study protocol was approved by the National Institutes of Biomedical Innovation, Health and Nutrition (DS18-086, DS18-086R1-DS18-086R21).

Human Mesothelioma Cell Line (JMN-1B)

Human mesothelioma cell line (JMN-1B) was kindly provided by Dr. Kunio Matsumoto, Osaka University, and were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS).

Intraperitoneal Administration of AHCC^® and Injection of JMN-1B Cells

AHCC^® (0.2 mg/g BW) and 0.9% saline were i.p. injected into three and three 12-week-old B6-scid male mice, respectively, 1 and 2 d before transplantation. JMN-1B cells (initial 4.6 × 10⁵) were i.p. injected into 6 mice (3 for AHCC^® treatment, 3 for Control). The tumor weights were measured after 83 d of injection.

Combination of Intraperitoneal and Oral Administration of AHCC^® and Injection with JMN-1B Cells

AHCC^® (0.2 mg/g BW) and 0.9% saline were injected i.p. into four and three 12-week-old B6-scid female mice, respectively. The mice were given 2.0% AHCC^® in the feed, after 5 and 8 d of intraperitoneal injection of AHCC^®, JMN-1B cells (initial 2.6 × 10⁵) were i.p. injected into those mice treated with AHCC^® and 0.9% saline. The weight of the intraperitoneal tumors was measured after 63 d of JMN-1B cell injection.

Intraperitoneal Administration of BCG and AHCC^® and Transplantation of Renal Cancer PDX

BCG (10 µg/g BW) (Lot: KH209, Japan BCG Laboratory, Tokyo, Japan) or AHCC^® (0.01 mg/g BW) was i.p. injected into C3H/HeJ-scid male mice 1 and 4 d before transplantation. Fifteen (BCG: 5 mice, AHCC^®: 5 mice, Control: 5 mice, with 5–8 weeks of age) mice were anesthetized and renal cancer PDX tissue pieces were subcutaneously transplanted into the back (both right and left sides) of the mice.

Combination of Intraperitoneal and Oral Administration of AHCC^® and Transplantation of Renal Cancer PDX

AHCC^® (0.05 mg/g BW) was i.p. injected into five C3H/HeJ-scid male mice 4 d before transplantation. At the same time, AHCC^® (0.5%) in drinking water was given to the AHCC^® group. Ten (AHCC^®: 5 mice, Control: 5 mice, with 15–22 weeks of age) C3H/HeJ-scid mice were anesthetized and renal cancer PDX tissue pieces were subcutaneously transplanted into the back (both right and left sides) of the mice.

Oral Administration of AHCC^® and Transplantation of Invasive Ductal Carcinoma PDX

Sixteen (0.5% AHCC^®: 6 mice, Control: 10 mice, with 19–27 weeks of age) C3H/HeJ-scid female mice were anesthetized and invasive ductal carcinoma PDX tissue pieces were subcutaneously transplanted into the back (both right and left sides) of the mice, then feed containing AHCC^® (0.5%) were given to the AHCC^® group. In another experiment, ten (2.0% AHCC^®: 5 mice, Control: 5 mice, with 11–20 weeks of age) C3H/HeJ-scid mice were anesthetized and invasive ductal carcinoma PDX tissue pieces were subcutaneously transplanted into the back (both right and left sides) of the mice. At the same time, feed containing AHCC^® (2.0%) was given to the AHCC^® group.

Oral Administration of AHCC^® and Transplantation of Non-invasive Ductal Carcinoma PDX

Fifteen (0.5% AHCC^®: 5 mice, 2.0% AHCC^®: 5 mice, Control: 5 mice, with 9–14 weeks of age) C3H/HeJ-scid female mice were anesthetized and pieces of non-invasive ductal carcinoma PDX tissue pieces were subcutaneously transplanted into the back (both right and left sides) of the mice. At the same time, feed containing AHCC^® (0.5 and 2.0%) was given to the AHCC^® group.

Statistical Analysis

All the data are shown as means and standard errors. Statistical analyses for the comparisons between two groups were performed by values at each time point with student unpaired t-test after confirming equal variances in F-test, using Microsoft Excel. For the comparisons between three groups, statistical analyses were performed at each time point by non-parametric ANOVA (Kruskal–Wallis) with Dunn’s post-hoc test using the Graphpad Prism 10 software package. A p value less than 0.05 was defined as statistically significant.

RESULTS

A trial examination was carried out whether AHCC^®, a functional food showed tumor growth inhibition in Super SCID-mice, using mesothelioma cell line (JMN-1B).

Effects of Intraperitoneal Administration of AHCC^® on the Growth of Mesothelioma Cells

JMN-1B cell growth in the intraperitoneal caused a significant number of cell masses that were to be measured by weight. An intraperitoneal pre-treatment of AHCC^® was found to inhibit the growth of JMN-1B cells with a decrease in the total intraperitoneal tumor weight, although this was not significant by t-test due to the insufficient number of mice (Control: 3, AHCC^®: 3) (Fig. 1).

Fig. 1. The Inhibitory Effect of Intraperitoneal Administration (i.p.) with AHCC^® on the Growth of JMN-1B Cells

AHCC^® (0.2 mg/g BW) and 0.9% saline were intraperitoneally injected into three and three 12-week-old B6-scid male mice, respectively, 1 and 2 d before transplantation. JMN-1B cells (initial 4.6 × 10⁵) were intraperitoneally injected into B6 scid mice. After 83 d of injection, the tumor weight was measured. Error bars represent the standard error of the mean.

Effects of Combination with Intraperitoneal and Oral Administration of AHCC^® in Mesothelioma Cells

The growth inhibitory effect of the intraperitoneal and oral (2.0% AHCC^® in feed) administration of AHCC^® to JMN-1B cells were studied. Although there was no significant difference, there was a decrease in the total intraperitoneal tumor weight in the AHCC^® treated group (Fig. 2).

Fig. 2. The Inhibitory Effect of Intraperitoneal Administration (i.p.) Combined with Oral Administration (p.o.) of AHCC^® on the Growth of JMN-1B Cells

AHCC^® (0.2 mg/g BW) and 0.9% saline were intraperitoneally injected into four and three 12-week-old B6-scid female mice respectively, 5 and 8 d before transplantation. JMN-1B cells (initial 2.6 × 10⁵) were intraperitoneally injected into B6-scid mice. After that, 2.0% AHCC^® in feed was given orally to those mice. After 63 d of injection, tumor weights were measured. Error bars represent the standard error of the mean.

Effect of Intraperitoneal Administration of BCG and AHCC^® on the Growth of Renal Cancer PDX

To investigate the inhibitory effect on the growth of renal cancer PDX by BCG, an enhancer of immune system by macrophage activation, a comparative study with administration of BCG and AHCC^® were conducted. In BCG or AHCC^® treated mice, tendency of tumor growth inhibition was shown but no significant difference (Fig. 3). In AHCC^® treated mice, mice were healthy appearance. In contrast, in control and BCG treated mice, unhealthy lusterless furs were observed in appearance.

Fig. 3. The Inhibitory Effect of Intraperitoneal Administration with BCG and AHCC^® on the Growth of Renal Cancer PDX

BCG and AHCC^® were intraperitoneally injected into five male C3H/HeJ-scid mice 1 and 4 d before transplantation, respectively. The tumor sizes were measured once a week. Error bars represent the standard error of the mean. Multiple group Comparisons were performed by non-parametric ANOVA (Kruskal–Wallis) with Dunn’s post-hoc test. BCG; BCG (10 µg/g BW) treatment group. AHCC^®; AHCC^® (0.01 mg/g BW) treatment group. Control; non-treatment control group.

Combination of Intraperitoneal and Oral Administration of AHCC^® Drinking Water to Renal Cancer PDX

Compared to the non-treatment mice, the AHCC^® treatment significantly inhibited the proliferation of renal cancer PDX from 48 to 83 d after transplantation (at day 48: p = 0.00202, day 55: p = 0.0000111, day 62: p = 0.000128, day 69: p = 0.00865, day 76: p = 0.0249, day 83: p = 0.0449) (Fig. 4).

Fig. 4. The Inhibitory Effect of AHCC^® by Combination of Intraperitoneal and Oral Administration with AHCC^® on the Growth of Renal Cancer PDX

AHCC^® were intraperitoneally injected (0.05 mg/g BW) into five male C3H/HeJ-scid mice 4 d before transplantation. At the same time as transplantation of renal cancer PDX, AHCC^® (0.5%) water was given to the AHCC^® group. The tumor sizes were measured once a week. Error bars represent the standard error of the mean. AHCC^®; combined administration of AHCC^® by i.p. (0.05 mg/g BW) and 0.5% AHCC^® drinking water. Control; non-treatment group. significant differrence: **; p < 0.01, *; p < 0.05 by t-test.

Effect of Oral Administration of AHCC^® in Feed on the Growth of Invasive Ductal Carcinoma PDX

The growth inhibition of invasive ductal carcinoma PDX by the 0.5% AHCC^® administration orally in feed did not show significantly. Nevertheless, 0.5% AHCC^® treatment showed a trend toward this ductal carcinoma PDX growth inhibition compared to that of non-treated PDX. This tendency was appeared by mean tumor volume measured from days 22 to 63 (Fig. 5).

Fig. 5. The Inhibitory Effect of Oral Administration with 0.5% AHCC^® in Feed on the Growth of Invasive Ductal Carcinoma PDX

0.5% AHCC^®; AHCC^® treatment group. Control; non-treatment group. Invasive ductal carcinoma PDX tissue pieces were transplanted in sixteen (0.5% AHCC^® group: 6 mice, Control group: 10 mice) female C3H/HeJ-scid mice. At the same time, AHCC^® (0.5%) in feed were given to the AHCC^® group. The tumor sizes were measured once a week. Error bars represent the standard error of the mean.

Tumor volume in mice with 2.0% AHCC^® treated orally were apparently smaller than that in control mice with increasing the day of tumor volume measurement from days 48 to 77 (Fig. 6). Namely, tumor growth inhibition by AHCC^® administration showed a tendency. In this period, a significant difference in tumor volume between that in mice with AHCC^® treated and in control was only at day 70 (p = 0.038) (Fig. 6).

Fig. 6. The Inhibitory Effect of the Oral Administration with 2.0% AHCC^® in Feed on the Growth of Invasive Ductal Carcinoma PDX

Invasive ductal carcinoma PDX tissue pieces were transplanted in 10 (2.0% AHCC^® group: 5 mice, Control group: 5 mice) female C3H/HeJ-scid mice, then AHCC^® (2.0%) in the feed was given to the AHCC^® group. The tumor sizes were measured once a week. Error bars represent the standard error of the mean. *; significant difference p < 0.05 by t-test.

Effect of Oral Administration with 0.5 and 2.0% AHCC^® in Feed on the Growth of Non-invasive Ductal Carcinoma PDX

To investigate dose dependency of AHCC^®, two different concentrations (0.5 and 2.0%) of AHCC^® in feed were given to SCID mice with non-invasive ductal carcinoma PDX. No difference in two different doses for the tumor PDX growth with AHCC^® administration was observed (Fig. 7). Significant inhibitory effect on this cancer PDX growth was found in 2.0% AHCC^® administrated mice only at day 14 (p = 0.0064). The administration of 0.5 and 2.0% AHCC^® tended to inhibit the growth of the non-invasive ductal carcinoma PDX (Fig. 7) as was similarly shown in the effects on the growth invasive ductal carcinoma PDX (Figs. 5, 6).

Fig. 7. The Inhibitory Effect of the Oral Administration with 0.5% and 2.0% AHCC^® in Feed on the Growth of Non-Invasive Ductal Carcinoma PDX

Pieces of non-invasive ductal carcinoma PDX were transplanted in fifteen female C3H/HeJ-scid mice, then they were divided into three groups, AHCC^® of 2.0%, 0.5% and Control group (each no of mice were 5). The tumor sizes were measured once a week. Error bars represent the standard error of the mean.0.5% AHCC^® and 2.0% AHCC^®; 0.5 and 2.0% AHCC^® in feed group, respectively. Control; non-treatment control group. *; significant difference for 2.0% AHCC^® p < 0.05 by non-parametric ANOVA (Kruskal–Wallis), followed by Dunn’s post-hoc test.

DISCUSSION

In Fig. 1, the intraperitoneal administration of AHCC^® to the mesothelioma cell lines tended to inhibit its cell growth, although the difference in AHCC^® treatment and control was not significant. The combination of oral administration also demonstrated similar results (Fig. 2). Since AHCC^® has been reported to increase the activity of NK cells and macrophages,^17,23) this growth inhibition might be attributed to the intraperitoneal activity of these mouse-derived immune cells.

The intraperitoneal pre-administration of AHCC^® (0.01 mg/g BW) or BCG (10 µg/g BW) to C3H/HeJ-scid mice implanting renal cancer PDX tended to suppress the growth of the cancer PDX (Fig. 3). One of the reasons for the tendency of suppression on the growth of renal cancer PDX by BCG and AHCC^® administration might be the administration methods for these two reagents. Since BCG would weaken the mice due to the side effect of BCG, BCG was given to the mice i.p. before PDX transplantation, and same method was adopted for the administration of AHCC^® as match to that of BCG. It might be too short time by this administration method to examine the inhibitory effects by two reagents. Generally, in pre-clinical study, anti-cancer reagents were administrated to SCID mice after transplantation of tumor PDX, and in clinical practice, AHCC^® were given during cancer growth in a patient. Difference in appearance for the tumor bearing mice treated by AHCC^®, BCG and AHCC^® un-treated control mice were observed. AHCC^® treated mice were healthy appearance, in contrast to the control and BCG-treated mice in which mice were unhealthy lusterless furs. This result for the appearance of the tumor PDX bearing mice administrated by AHCC^® may reflect the clinical findings in humans where AHCC^® improved palliate the medical condition of weight loss and poor physical symptoms caused by the cancer or anti-cancer drug treatment.^25,30)

A significant growth inhibition of renal cancer PDX was observed with combination of intraperitoneal and oral administration as AHCC^® in drinking water. In this experiment, AHCC^® administration started i.p. 4 d before transplantation of the PDX and the oral administration continued after transplantation until the end of the experiment (Fig. 4).

The effects of AHCC^® on the growth of breast cancer, which is the highest incidence among cancers in women, worldwide and in Japan,³²⁾ were also evaluated using two types of breast cancer PDX. The growth suppression effect on the growth of aggressive invasive ductal carcinoma PDX was examined. The tendency of growth suppression of this PDX was found by 0.5% AHCC^® administration in the feed at the same time as this PDX transplantation, although no significant difference by t-test (Fig. 5). A similar experiment was performed with 2.0% AHCC^® administration in feed to evaluate the effect of dose. A significant tumor growth inhibitory effect by AHCC^® was observed at only day 70 (Fig. 6). No large difference was found between 0.5 and 2.0% AHCC^® administration for suppression of the invasive ductal carcinoma PDX growth (Figs. 5, 6). Two different AHCC^® concentrations in feed (0.5 and 2.0%) were prepared to investigate the administration dose dependency of AHCC^® on the efficacy of growth suppression in non-invasive ductal carcinoma PDX (Fig. 7). The administration of AHCC^® at a concentration of 2.0% had a significant inhibitory effect on the PDX growth at day 14, while no significant differences by concentration (Fig. 7). Tendency of growth suppression of non-invasive breast cancer PDX by administration with 0.5 and 2.0% AHCC^® in feed were again shown by the growth curve (Fig. 7). Although the minimum concentration in both invasive and non-invasive ductal carcinoma PDX experiments was set at 0.5%, the lower concentrations might be effective. These invasive and non-invasive ductal carcinoma PDX showed a rapid growth rate compared to the growth rate of this renal cancer (Figs. 4–7). This non-invasive ductal carcinoma PDX were growing to nearly 2000 mm³, the ethically acceptable upper limit for experimental animals, in approx. one month. Thus, the period of the study is limited to approx. 30 d for this kind of rapid growth PDX, and it is not easy for the reagent having a weak suppressibility for tumor growth like AHCC^® to obtain significant differences in Super SCID-PDX model. Longer period of experiment like in the renal cancer PDX, made a significant efficiency of reagents on tumor PDX growth (Fig. 4). These results indicated that the growth inhibitory effects of AHCC^® by the methods we applied in the experiments partly depended on a characteristic of the cancer, probably the growth rate as one of the reasons, and it may require time for the immune response by the AHCC^® administration during the trial period. Therefore, in clinical practice, AHCC^® should be used in combination with surgery, radiotherapy, or anti-cancer therapy to slow the tumor growth as much as possible, rather than an AHCC^® treatment alone. Other reason that the significant suppression of the renal cancer PDX growth by AHCC^® compared to the lack of significant growth suppression of the two types of breast cancer PDX is speculated as the difference in AHCC^® administration route, dose and timing before PDX implantation and AHCC^® water intake to SCID mice. In the experiment of renal cancer in PDX-SCID model, AHCC^® was administrated i.p. 4 d before PDX transplantation, and orally as drinking water, and the oral administration (drinking water) continued to the end of experiment. Whereas, in the experiments for the studies on the growth inhibitory effects by AHCC^® in two types of breast cancer PDX, AHCC^® were given orally in feed at the same time of tumor PDX transplantation. Another reason for the failure of significant suppression on the growth of two types of breast cancer PDX by AHCC^® might be due to the large variation in size for tumor PDX at each time point during tumor growth in SCID mice. The large variation in tumor size would be diminished if the SCID mice having tumor PDX with approximately same size are prepared. Namely, administration of test reagents should be started when the transplanted PDXs are grow to make approx. same size in PDX volume. By this method, AHCC^® treatment is expected to suppress significantly for the growth of two types of breast cancer PDX.^10–12)

The amount of AHCC^® intake in feed for SCID mouse in this experiment was evaluated. A mouse (weight approx. 26 g) used for this experiment intake an average of 2.1 g/d of food containing 0.5% AHCC^®, namely, a mouse had 10.5 mg/d of AHCC^® was ingested. This daily intake value in a mouse was equivalent to approx. 2 g/d of AHCC^® for an adult human weighing 60 kg based on the calculation by body surface.³³⁾ This estimated value, 2 g/d/person for AHCC^® intake in human is quite close to the AHCC^® intake value, approx. 3 g/d which is used in clinical practice.

Compared to cell line models, in the SCID-PDX models, tumor PDX is more similar to human cancer tissue, since this model uses grafts by transplantation of original human tissues,³⁴⁾ and the tumor PDX retains tissue components as tumor-infiltrating lymphocytes (TILs) and stromal tissue other than cancer cells. TILs in tumor PDX might be one of the reasons for the anti-tumor activities of AHCC^®.³⁵⁾

Recently, some researchers have attempted to transfuse SCID mice with human peripheral blood mononuclear cells in order to create a mouse model (humanized mouse) that is close to humans. However, since there is a concern about the incidence of graft versus host disease (GVHD) in this model mice due to peripheral blood mononuclear cell engraftment,³⁶⁾ it is not proper as a standard evaluation method at the moment. Organoids and spheroids have been highlighted and attempted to create homogeneous tumor tissue, however these are morphologically very different from human tissue.^37,38) Thus, PDX remains very meaningful in the preclinical studies of drug development. The experiments demonstrated that PDX has a potential to be a useful model for the functional foods that stimulate the immune system, such as AHCC^®.

Acknowledgments

We would like to acknowledge for providing the human tissues to the Osaka Police Hospital and for providing JMN-1B cell line to Dr. Kunio Matsumoto. We thank to Dr. Takahiro Tougan for his guidance on statistical analysis and for his critical discussion on statistical results, and to Dr. Hiroo Nakajima for his guidance on this manuscript.

Funding

This study was supported by the Human Science Foundation (KHD1223) of the Ministry of Health, Labor and Welfare of Japan and a Grant from Amino Up Co., Ltd. (Sapporo, Japan).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Zhang X, Claerhout S, Prat A, et al. A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models. Cancer Res., 73, 4885–4897 (2013).
• 2) Ohgidani M, Ichihara H, Goto K, Matsumoto Y, Ueoka R. Anticancer effects of residual powder from Barley–Shochu distillation remnants against the orthotopic xenograft mouse models of hepatocellular carcinoma in vivo. Biol. Pharm. Bull., 35, 984–987 (2012).
• 3) Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse. Nature, 301, 527–530 (1983).
• 4) Tsujimura A, Fukuhara S, Soda T, Takezawa K, Kiuchi H, Takao T, Miyagawa Y, Nonomura N, Adachi S, Tokita Y, Nomura T. Histologic evaluation of human benign prostatic hyperplasia treated by dutasteride: a study by xenograft model with improved severe combined immunodeficient mice. Urology, 85, 274.e1–8 (2015).
• 5) Okada S, Vaeteewoottacharn K, Kariya R. Application of highly immunocompromised mice for the establishment of patient-derived xenograft (PDX) models. Cells., 8, 889 (2019).
• 6) Abdolahi S, Ghazvinian Z, Muhammadnejad S, Saleh M, Asadzadeh Aghdaei H, Baghaei K. Patient-derived xenograft (PDX) models, applications and challenges in cancer research. J. Transl. Med., 20, 206 (2022).
• 7) Adachi S, Ryo H, Hongyo T, Nakajima H, Tsuboi-Kikuya R, Tokita Y, Matsuzuka F, Hiramatsu K, Fujikawa K, Itoh T, Nomura T. Effects of fission neutrons on human thyroid tissues maintained in SCID mice. Mutat. Res., 696, 107–113 (2010).
• 8) Nomura T, Hongyo T, Nakajima H, Li LY, Syaifudin M, Adachi S, Ryo H, Baskar R, Fukuda K, Oka Y, Sugiyama H, Matsuzuka F. Differential radiation sensitivity to morphological, functional and molecular changes of human thyroid tissues and bone marrow cells maintained in SCID mice. Mutat. Res., 657, 68–76 (2008).
• 9) Nomura T, Nakajima H, Hongyo T, Taniguchi E, Fukuda K, Li LY, Kurooka M, Sutoh K, Hande PM, Kawaguchi T, Ueda M, Takatera H. Induction of cancer, actinic keratosis, and specific p53 mutations by UVB light in human skin maintained in severe combined immunodeficient mice. Cancer Res., 57, 2081–2084 (1997).
• 10) Koganemaru S, Kuboki Y, Koga Y, Kojima T, Yamauchi M, Maeda N, Kagari T, Hirotani K, Yasunaga M, Matsumura Y, Doi T. U3-1402, a novel HER3-targeting antibody-drug conjugate, for the treatment of colorectal cancer. Mol. Cancer Ther., 18, 2043–2050 (2019).
• 11) Pharmaceutical Interview Forms of enhertu^® for intravenous drip infusion, Japan standard commodity classification 874291, Nov. (2022).
• 12) Iida K, Abdelhamid Ahmed AH, Nagatsuma AK, et al. Identification and therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumors, with DS-6157a, a first-in-class antibody-drug conjugate. Cancer Discov., 11, 1508–1523 (2021).
• 13) Lindequist U, Niedermeyer TH, Jülich WD. The pharmacological potential of mushrooms. Evid. Based Complement Alternat. Med., 2, 285–299 (2005).
• 14) Ritz BW. Supplementation with active hexose correlated compound increases survival following infectious challenge in mice. Nutr. Rev., 66, 526–531 (2008).
• 15) Smith JA, Gaikwad AA, Mathew L, Rech B, Faro JP, Lucci JA 3rd, Bai Y, Olsen RJ, Byrd TT. AHCC^® supplementation to support immune function to clear persistent human papillomavirus infections. Front. Oncol., 12, 881902 (2022).
• 16) Mascaraque C, Suárez MD, Zarzuelo A, Sánchez de Medina F, Martínez-Augustin O. Active hexose correlated compound exerts therapeutic effects in lymphocyte driven colitis. Mol. Nutr. Food Res., 58, 2379–2382 (2014).
• 17) Matsushita K, Kuramitsu Y, Ohiro Y, Obara M, Kobayashi M, Li YQ, Hosokawa M. Combination therapy of active hexose correlated compound plus UFT significantly reduces the metastasis of rat mammary adenocarcinoma. Anticancer Drugs, 9, 343–350 (1998).
• 18) Takanari J, Sato A, Waki H, Miyazaki S, Uebaba K, Hisajima T. Effects of AHCC^® on immune and stress responses in healthy individuals. J. Evid. Based Integr. Med., 23, 2156587218756511 (2018).
• 19) de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat. Rev. Cancer, 6, 24–37 (2006).
• 20) Kumar P, Tyagi R, Das G, Bhaskar S. Mycobacterium indicus pranii and Mycobacterium bovis BCG lead to differential macrophage activation in Toll-like receptor-dependent manner. Immunology, 143, 258–268 (2014).
• 21) Rhoades E, Hsu F-F, Torrelles JB, Turk J, Chatterjee D, Russell DG. Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCG. Mol. Microbiol., 48, 875–888 (2003).
• 22) Galligioni E, Francini M, Quaia M, Carbone A, Spada A, Sacco C, Favaro D, Santarosa M, Carmignani G, Di Donna D, Mazza G, Viggiano S, Fiaccavento G, Dal Bo V, Talamini R. Randomized study of adjuvant immunotherapy with autologous tumor cells and BCG in renal cancer. Ann. N. Y. Acad. Sci., 690, 367–369 (1993).
• 23) Gao Y, Zhang D, Sun B, Fujii H, Kosuna K, Yin Z. Active hexose correlated compound enhances tumor surveillance through regulating both innate and adaptive immune responses. Cancer Immunol. Immunother., 55, 1258–1266 (2006).
• 24) Koch GE, Smelser WW, Chang SS. Side effects of intravesical BCG and chemotherapy for bladder cancer: what they are and how to manage them. Urology, 149, 11–20 (2021).
• 25) Matsui Y, Uhara J, Satoi S, Kaibori M, Yamada H, Kitade H, Imamura A, Takai S, Kawaguchi Y, Kwon AH, Kamiyama Y. Improved prognosis of postoperative hepatocellular carcinoma patients when treated with functional foods: a prospective cohort study. J. Hepatol., 37, 78–86 (2002).
• 26) Yanagimoto H, Satoi S, Yamamoto T, Hirooka S, Yamaki S, Kotsuka M, Ryota H, Michiura T, Inoue K, Matsui Y, Tsuta K, Kon M. Alleviating effect of active hexose correlated compound (AHCC^®) on chemotherapy-related adverse events in patients with unresectable pancreatic ductal adenocarcinoma. Nutr. Cancer, 68, 234–240 (2016).
• 27) Hirose A, Sato E, Fujii H, Sun B, Nishioka H, Aruoma OI. The influence of active hexose correlated compound (AHCC) on cisplatin-evoked chemotherapeutic and side effects in tumor-bearing mice. Toxicol. Appl. Pharmacol., 222, 152–158 (2007).
• 28) Ishizuka R, Fujii H, Miura T, Fukuchi Y, Tajima K. Personalized cancer therapy for stage IV non-small cell lung cancer: combined use of active hexose correlated compound and genistein concentrated polysaccharide. Personalized Medicine Universe, 1, 39–44 (2012).
• 29) Parida DK, Wakame K, Nomura T. Integrating complimentary and alternative medicine in form of active hexose co-related compound (AHCC) in the management of head & neck cancer patients. International Journal of Clinical Medicine., 2, 588–592 (2011).
• 30) Kamiyama T, Orimo T, Wakayama K, Kakisaka T, Shimada S, Nagatsu A, Asahi Y, Aiyama T, Kamachi H, Taketomi A. Preventing recurrence of hepatocellular carcinoma after curative hepatectomy with active hexose-correlated compound derived from Lentinula edodes mycelia. Integr. Cancer Ther., 21, 15347354211073066 (2022).
• 31) Ye SF, Ichimura K, Wakame K, Ohe M. Suppressive effects of active hexose correlated compound on the increased activity of hepatic and renal ornithine decarboxylase induced by oxidative stress. Life Sci., 74, 593–602 (2003).
• 32) Iwasaki M, Tsugane S. Risk factors for breast cancer: epidemiological evidence from Japanese studies. Cancer Sci., 102, 1607–1614 (2011).
• 33) Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm., 7, 27–31 (2016).
• 34) Hidalgo M, Amant F, Biankin AV, Budinská E, Byrne AT, Caldas C, Clarke RB, de Jong S, Jonkers J, Mælandsmo GM, Roman-Roman S, Seoane J, Trusolino L, Villanueva A. Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov., 4, 998–1013 (2014).
• 35) Bai Z, Zhou Y, Ye Z, Xiong J, Lan H, Wang F. Tumor-infiltrating lymphocytes in colorectal cancer: the fundamental indication and application on immunotherapy. Front. Immunol., 12, 808964 (2022).
• 36) Kanikarla Marie P, Sorokin AV, Bitner LA, Aden R, Lam M, Manyam G, Woods MN, Anderson A, Capasso A, Fowlkes N, Overman MJ, Menter DG, Kopetz S. Autologous humanized mouse models to study combination and single-agent immunotherapy for colorectal cancer patient-derived xenografts. Front Oncol., 12, 994333 (2022).
• 37) Onozato D, Akagawa T, Kida Y, Ogawa I, Hashita T, Iwao T, Matsunaga T. Application of human induced pluripotent stem cell-derived intestinal organoids as a model of epithelial damage and fibrosis in inflammatory bowel disease. Biol. Pharm. Bull., 43, 1088–1095 (2020).
• 38) Ueno Y, Taniguchi H. Prospects for drug evaluation using human cancer organoid technology. Organ Biology, 27, 119–124 (2020).